Streamlined high-throughput cloning protocol to generate arrayed mutant libraries.

Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format. The protocol allows most molecular biology labs to clone 96 mutants (from PCR to sequence ready plasmid) in 3 days.

Performance of medical students in a nontraditional rural clinical program, 1998-99 through 2003-04.

PURPOSE: To compare knowledge acquisition as measured by test scores for students in nontraditional clinical clerkships to scores for students in traditional urban hospital-based clerkships. Interdisciplinary and continuity-of-care clerkships in rural areas are the focus of the study. METHOD: All the students' Medical College Admission Test (MCAT) scores, National Board of Medical Examiners (NBME) subject exam scores, and United States Medical Licensing Examination (USMLE) Step 1 and Step 2 scores over a six-year period, 1998-99 to 2003-04, were compared for third-year students in nontraditional and traditional clerkships at the University of North Dakota School of Medicine and Health Sciences. Cohorts were 29 students in our Rural Opportunities in Medical Education (ROME) program and 296 students in traditional third-year clerkships. NBME subject exam scores were those in pediatrics, internal medicine, surgery, and obstetrics and gynecology. The exam used for family medicine is not standardized to national standards, but controlled within the Department of Family Medicine. MCAT and USMLE Step 1 scores were used as a means of controlling for prior academic achievement and ability. RESULTS: There were no significant differences (p > or = .05) in MCAT scores, Step 1 scores, subject exam scores, or Step 2 scores between the two groups. In contrast, students from ROME scored higher (p < or = .05) on the internal medicine clinical preceptor assessments than did students from the traditional track. CONCLUSIONS: These findings suggest that students in remote, rural, longitudinal, integrated learning environments can attain fund-of-knowledge scores comparable to the scores of students in traditional clerkships, and may, as in this study, receive higher ratings for clinical proficiency.

High-yield expression of isotopically labeled peptides for use in NMR studies.

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C.

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.